Tumor-targeting Salmonella typhimurium A1-R combined with recombinant methioninase and cisplatinum eradicates an osteosarcoma cisplatinum-resistant lung metastasis in a patient-derived orthotopic xenograft (PDOX) mouse model: decoy, trap and kill chemotherapy moves toward the clinic

ABSTRACT

In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G₂, and chemotherapy. The PDOX models were randomized into the following groups 14 days after implantation: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.

KEYWORDS:

• osteosarcoma
• metastasis
• lung
• PDOX
• resistance
• Salmonella typhimurium A1-R
• decoy
• methioninase
• trap
• cisplatinum
• kill

Introduction

Metastatic osteosarcoma is a recalcitrant disease with a less than 20% long-term survival rate which has not improved for many years [1–7].

In order to develop precision individualized therapy for metastatic ostesosarcoma, we previously established a patient-derived orthotopic xenogrraft (PDOX) of a lung-metastasis from an osteosarcoma of a patient who failed CDDP therapy. Temozolomide (TEM) and trabectedin (TRAB), but not CDDP, significantly inhibited tumor volume compared to untreated control in the PDOX model. This osteosarcoma PDOX model identified potentially, highly-effective drugs for this recalcitrant disease, while accurately maintaining the CDDP resistance of the tumor in the patient [8].

We also previously reported that a subcutaneous mouse model of this CDDP-resistant ostoeosarcoma was sensitive to tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) [9].

We also previously showed recombinant methioninase (rMETase) effectively reduced tumor growth of a PDOX model of Ewing's sarcoma compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group [10].

Tumor-targeting S. typhimurium A1-R decoyed chemo-resistant quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M. When the cancer cells were subsequently treated with rMETase, they were selectively trapped in S/G₂. We showed using sequential treatment of tumors in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle and rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, that chemotherapy could eradicate tumors in mouse models of human stomach cancer. These results demonstrated a new praradigm of “decoy, trap and shoot (kill)” chemotherapy [11].

In the present study, we show that sequential treatment of the chemotherapy-resistant osteosarcoma lung metastasis in a PDOX mouse model with S. typhimurium A1-R, rMETase and CDDP could eradicate the tumor.

Results and discussion

Sequential treatment of the chemotherapy-resistant osteosarcoma lung metastasis PDOX mouse model with S. typhimurium A1-R, rMETase and CDDP

All treatments but CDDP significantly inhibited tumor growth compared to the untreated control on day 14 after initiation (Figures 1 and 2). Tumor volume at day 14 were the following: control (G1): 201±91 mm³; CDDP (G2): 174±108 mm³; rMETase (G3): 79±41 mm³; S. typhimurium A1-R (G4): 74±18 mm³; S. typhimurium A1-R+ rMETase (G5) 44±15 mm³; S. typhimurium A1-R+rMETase+CDDP (G6) 15±8 mm³. Control vs. CDDP (p = 0.586); control vs. rMETase (p = 0.002); control vs. S. typhimurium A1-R (p = 0.002); control vs. S. typhimurium A1-R+rMETase (p = 0.0004); control vs. S. typhimurium A1-R+rMETase+CDDP (p = 0.0001); CDDP vs. rMETase (p = 0.045); CDDP vs. S. typhimurium A1-R (p = 0.033); CDDP vs. S. typhimurium A1-R+rMETase (p = 0.011); CDDP vs. S. typhimurium A1-R+rMETase+CDDP (p = 0.004); rMETase vs. S. typhimurium A1-R (p = 0.719); rMETase vs. S. typhimurium A1-R+rMETase (p = 0.048); rMETase vs. S. typhimurium A1-R+rMETase+CDDP (p = 0.003); S. typhimurium A1-R vs. S. typhimurium A1-R+rMETase (p = 0.003); S. typhimurium A1-R vs. S. typhimurium A1-R+rMETase+CDDP (p<0.0001); S. typhimurium A1-R+rMETase vs. S. typhimurium A1-R+rMETase+CDDP (p = 0.0004) (Figures 3 and 4). There were no animal deaths in any group. The body weight of treated mice was not significantly different in any group (Figure 5).

Figure 1. Establishment of osteosarcoma lung metastaswas PDOX model. A) A skin incision was made on the left chest wall. B) Chest muscles were separated and an intercostal incision in the chest wall was made, and the chest wall was opened. C) The left lung was taken up and tumor fragments were sewn into the lower lung with one suture. D) The incision in the chest wall was closed with a 6-0 surgical suture. E) An intrathoracic puncture was made to withdraw the remaining air in the chest cavity.

Figure 2. Treatment schema.

Figure 3. Quantitative in vivo antitumor efficacy of monotherapy of CDDP, rMETase, S. typhimurium A1-R, S. typhimurium A1-R + rMETase and S. typhimurium A1-R + rMETase + CDDP on the lung metastatic osteosarcoma PDOX. Please see Figure 2 for treatment schema. Tumor volume was measured at day 14 at necropsy. N = 8 mice/group. *p<0.005, **p<0.001

Figure 4. Representative photos of treated and untreated osteosarcoma lung-metastatic PDOX models.

Figure 5. Effect of treatments on osteosarcoma lung metastasis PDOX on mouse body weight. Bar graph shows relative body weight in each treatment group at pre- and post-treatment relative to initial body weight. There were no significant differences between any of the treatment groups or the untreated groups.

Histology of the original tumor and implanted tumors

High power microscopy of the original patient tumor showed neoplastic chondroid matrix occupied by anaplastic cells. The tumor had hypercellular areas populated by anaplastic cells displaying nuclear pleomorphism, coarse and hyperchromatic chromatin and abundant mitotic figures (Figure 6A). High power microscopy of the untreated PDOX tumor showed solid and chondroblastic appearance similar to the patient original tumor with hypercellular areas filled with tumor cells displaying nuclear pleomorphism and mitotic figures (Figure 6B). The PDOX tumor treated with CDDP comprised viable cells without apparent necrosis or inflammatory changes and similar features compared to the untreated control (Figure 6C). The rMETase-treated tumor and S. typhimurium A1-R-treated tumor showed changes in sarcoma-cell shapes (Figure 6D and 6E). S. typhimurium A1-R combined with rMETase-treated tumor showed reduced cellularity (Figure 6F). The tumor treated with S. typhimurium A1-R combined with both rMETase and CDDP showed reduced cellularity and tumor necrosis (Figure 6G) [8].

Figure 6. Effect of treatment on osteosarcoma lung metastasis PDOX tumor histology. A) Hematoxylin and eosin (H&E)-stained section of the original patient lung metastasis. B) Untreated PDOX tumor. C) PDOX tumor treated with CDDP. D) PDOX tumor treated with rMETase. E) PDOX tumor treated with S. typhimurium A1-R. F) PDOX tumor treated with S. typhimurium A1-R combined with rMETase and G) PDOX tumor treated with S. typhimurium A1-R combined with both rMETase and CDDP. White scale bars: 80µm.

The present study was made possible by the use of a PDOX model which closely mimics the patient. Toward this goal, our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI), including breast [12], ovarian [13], lung [14], cervical [15,16], colon [17–19], stomach [20], pancreatic [21–25], melanoma [26–30], and sarcoma [31–40]. The PDOX model, developed by our laboratory over the past 30 years, has many advantages over subcutaneous-transplant models which are growing ectopically under the skin [41]. The PDOX model enables precise, individualized therapy, especially for recalcitrant diseases, for example, metastatic melanoma [26–30] or sarcoma [31–40] by matching the patient tumor to an effective drug identified with the PDOX models.

S. typhimurium A1-R may be a general therapeutic for cancer. S. typhimurium A1-R is auxotrophic for Leu–Arg, which prevents it from mounting a continuous infection in normal tissues. S. typhimurium A1-R inhibited or eradicate primary and metastatic tumors as monotherapy in nude-mouse models of major cancers [42], including prostate [43,44], breast [45–47], lung [48,49], pancreatic [23,50–53], ovarian [54,55], stomach [56], cervical cancer [57], glioma [58,59], as well as sarcoma [32,60], including osteosarcoma [61–63], all of which are highly aggressive tumor models.

rMETase may also be a general therapeutic for cancer since methionine dependence appears to be a general metabolic defect in cancer [11,64–77].

Previously-developed concepts and strategies of highly-selective tumor targeting can take advantage of molecular targeting of tumors, including tissue-selective therapy which focuses on unique differences between normal and tumor tissues [78–83].

Materials and methods

Animal care

Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4–6 weeks old, were used in this study. Animals were housed in a barrier facility on a high efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet. All animal studies were conducted with an AntiCancer Institutional Animal Care and Use Committee (IACUC)-protocol specifically approved for this study and in accordance with the principles and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. In order to minimize any suffering of the animals, anesthesia and analgesics were used for all surgical experiments. Animals were anesthetized by subcutaneous injection of a 0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate. The response of animals during surgery was monitored to ensure adequate depth of anesthesia. The animals were observed on a daily basis and humanely sacrificed by CO₂ inhalation when they met the following humane endpoint criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant body weight loss, difficulty breathing, rotational motion and body temperature drop [8].

Patient-derived tumor

The study was previously reviewed and approved by the UCLA Institutional Review Board (IRB #10-001857) before the study began. Written informed consent was previously obtained from the patient as part of the above-mentioned UCLA Institutional Review Board-approved protocol. A 16-year old patient with localized left distal femoral high grade osteosarcoma previously underwent CDDP based neoadjuvant chemotherapy and limb salvage with distal femoral replacement. The tumor necrosis rate of the primary tumor after cisplatin based chemotherapy was 70%. One year later, the osteosarcoma relapsed with three bilateral metachronous pulmonary metastases. The patient was treated with curative surgery at the Division of Surgical Oncology, University of California, Los Angeles (UCLA). The patient did not receive chemotherapy or radiotherapy prior to lung surgery [8].

Surgical orthotopic implantation (SOI) for establishment of PDOX model

The previously established osteosarcoma PDOX was further established in the lung of nude mice in a previous study [8]. After anesthesia, mice are put in a position of right lateral decubitus, with four limbs restrained. A 1.5 cm transverse incision of the skin was made in the left chest wall. Chest muscles were separated by sharp dissection and costal and intercostal muscles were exposed. A 0.8-1.0 cm intercostal incision between the sixth and seventh rib on the chest wall was made, and the chest wall was opened. The left lung was taken up with a forceps, and tumor fragments were sewn promptly into the lower lung with one suture. The lung was then returned into the chest cavity. The incision in the chest wall was closed by a 6-0 surgical suture (Ethilon, Ethicon, Inc., NJ, USA). The closed condition of the chest walls examined immediately, and if a leak existed, it was closed by additional sutures. After closing the chest wall, an intrathoracic puncture was made by using a 3-ml syringe and 25G 1/2 needle to withdraw the remaining air in the chest cavity. After the withdrawal of air, a completely inflated lung could be seen through the thin chest wall of the mouse. Then the skin and chest muscle were closed with a 6-0 surgical suture in one layer (Figure 1) [14].

Preparation and administration of S. typhimurium A1-R

GFP-expressing S. typhimurium A1-R (AntiCancer Inc.) were grown overnight on LB medium (Fisher Sci., Hanover Park, IL, USA) and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then diluted in PBS. For an intra-venous injection, a total of 5 × 10⁷ CFU S. typhimurium A1-R in 100 μl PBS was administered to each mouse [43–45].

rMETase production

The pAC-1 rMETase high expression clone was used for rMETase production. The fermentation procedure for host E.coli cells and the purification protocol for rMETase were the same as previously described: rMETase was purified by 3 different steps using columns of DEAE Sepharose FF and Sephacryl S-200HR, and ActiClean Etox, which is designed for eliminating endotoxin [77].

Treatment study design

The osteosaroma PDOX lung-metastasis models were randomized into the following groups 14 days after implantation (Figures 1 and 2): G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 10⁷ CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks) (Figure 2). Body weight was measured with a digital balance twice a week. 14 days after initiation of treatment, all mice were sacrificed and tumors in the lung were assessed. Tumor volume was calculated by following formula: Tumor volume (mm³) = length (mm) × width (mm) × width (mm) × 1/2. Data are presented as mean ± SD.

Histological examination

Fresh tumor samples were fixed in 10% formalin and embedded in paraffin before sectioning and staining. Tissue sections (3 μm) were deparaffinized in xylene and rehydrated in an ethanol series. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. Histological examination was performed with a BHS system microscope. Images were acquired with INFINITY ANALYZE software (Lumenera Corporation, Ottawa, Canada) [8].

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

National Cancer Institute (grant number CA213649).